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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 December 2012 to 16 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Physical state: White powder.
- Storage condition of test material: Controlled room temperature (15 - 25 ºC, below 70 RH %).

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM
L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium). During the treatments, the serum content of the medium was reduced to 5 (v/v) % (DMEM-5).
- Properly maintained: Yes, cell stocks were kept in a freezer at -80 ± 10°C. Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air.
- Periodically checked for Mycoplasma contamination: Yes.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
> Experiment without metabolic activation: 2000, 1000, 500, 250, 125, 62.5 and 31.25 µg/mL
> Experiment with metabolic activation: 4000, 2000, 1000, 500, 250, 125, 62.5 and 31.25 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: Based on the preliminary solubility test, no proper formulation of the test material could be made at 500, 250 or 100 mg/mL using Distilled water. However, a formulation at a concentration of 500 mg/mL using Dimethyl sulfoxide (DMSO) as vehicle was suitable for the test. As DMSO is compatible to the test system, it was selected as the vehicle for the study. The highest examined concentration in the preliminary test was 5000 µg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.

DURATION
- Preincubation period: 1-3 day old cultures were incubated without the test solution for approximately 24 hours at 37 °C in 10 mL of culture medium.
- Exposure duration:
> Assay 1: 3 hours with and without S9 mix,.
> Assay 2: 3 hours with and 20 hours without S9 mix.
- Fixation time (start of exposure up to harvest of cells):
> Assay 1: 20 hours.
> Assay 2: 28 hours.

SPINDLE INHIBITOR: Colchicine (0.2 µg/mL).
STAIN: 5% Giemsa solution.

NUMBER OF REPLICATIONS: Duplicate.

NUMBER OF CELLS EVALUATED: At least one hundred metaphases with 22 ± 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible, for a total of 200 metaphases per concentration. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

DETERMINATION OF CYTOTOXICITY
- Method: % Relative survival.

OTHER EXAMINATIONS:
- Polyploidy and endoreplication’s were scored.
- Other: Marked reductions in the numbers of cells on the slides were recorded if needed.

PRELIMINARY TEST:
- A preliminary test was performed to determined cytotoxicity. The test was performed as described for the main test; except single cultures were used and positive controls were not included. Two separate assays were performed, A and B. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-hour harvesting time.
- Concentrations: A total of eight test concentrations between 5000 and 39.06 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
- Evaluation: At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as % relative survival.
Evaluation criteria:
The assay is considered valid, if the following criteria are met:
> The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
> The positive controls induce increases in the aberration frequency, which are significant.

The test material is considered to have shown clastogenic activity in this study if all of the following criteria are met:
> Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
> The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
> The increases are statistically significant.
> The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

The test material is concluded to have given a negative response if no reproducible, statistically significant increases are observed.
Statistics:
For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed at concentrations of 500 µg/mL or greater
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
CHROMOSOME ABERRATION ASSAYS
None of the treatment concentrations caused a biologically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation. At 2000 µg/mL with metabolic activation in Assay 1, a marginal statistically significant increase was recorded, compared with the corresponding negative control which had no aberrant cells in either replicate. However, both replicates at 2000 µg/mL were observed to have 3 aberrant cells, which is well within the negative historical control range of 0-5.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No large changes were observed.
- Effects of osmolality: No large changes were observed.
- Water solubility:
> In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 2000-125 µg/mL concentration range (experiment without metabolic activation) or 4000-125 µg/mL concentration range (experiment with metabolic activation).
> In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium in the 2000-250 µg/mL concentration range (experiment without metabolic activation) or 4000-125 µg/mL concentration range (experiment with metabolic activation).

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the historical control range of the testing laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
> In Assay 1, marked cytotoxicity was observed at 2000, 1000 and 500 µg/mL without metabolic activation (relative survival values were 21, 28 and 50%, respectively) and at 4000 and 2000 µg/mL with metabolic activation (relative survival values were 49 and 49%, respectively).
> In Assay 2, cytotoxicity was also observed at 2000, 1000 and 500 µg/mL without metabolic activation (relative survival values were 25, 25 and 49%, respectively) and at 4000, 2000 and 1000 µg/mL with metabolic activation (relative survival values were 35, 42 and 43%, respectively).

ADDITIONAL INFORMATION ON POLYPLOID ABD ENDOREDUPLICATED METAPHASES:
Polyploid metaphases (1 - 2) were found in some cases in the negative (vehicle) control or test item treated samples in the performed experiments. Endoreduplicated metaphases (1 - 2) were found in some cases in the positive control or test item treated samples in the performed experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of Results Main Assay 1

Concentration (µg/mL)

Time of Treatment/Sampling

Relative Survival (%)#

Insolubility##

Mean % Aberrant Cells###

Without metabolic activation (-S9)

Negative control

3 h/20 h

100

-

0.5

2000

3 h/20 h

21

+

NE

1000

3 h/20 h

28

+

NE

500

3 h/20 h

50

+

0.0

250

3 h/20 h

64

+a

0.5

125

3 h/20 h

90

+a

0.0

62.5

3 h/20 h

90

-b

NE

31.25

3 h/20 h

96

-

NE

Positive Control

3 h/20 h

72

-

16.9***

With metabolic activation (+S9)

Negative control

3 h/20 h

100

-

0.0

4000

3 h/20 h

49

+

NE

2000

3 h/20 h

49

+

3.0*

1000

3 h/20 h

67

+

2.5

500

3 h/20 h

82

+

2.0

250

3 h/20 h

79

+a

NE

125

3 h/20 h

83

+a

NE

62.5

3 h/20 h

91

-b

NE

31.25

3 h/20 h

88

-

NE

Positive Control

3 h/20 h

57

-

93.8***

 

Table 2: Summary of Results Main Assay 2

Concentration (µg/mL)

Time of Treatment/Sampling

Relative Survival (%)#

Insolubility##

Mean % Aberrant Cells###

Without metabolic activation (-S9)

Negative control

20 h/28 h

100

-

2.0

2000

20 h/28 h

25

+

NE

1000

20 h/28 h

25

+

NE

500

20 h/28 h

49

+

4.0

250

20 h/28 h

78

+a

3.0

125

20 h/28 h

81

-b

1.0

62.5

20 h/28 h

91

-b

NE

31.25

20 h/28 h

87

-

NE

Positive Control

20 h/28 h

56

-

21.0***

With metabolic activation (+S9)

Negative control

3 h/28 h

100

-

3.5

4000

3 h/28 h

35

+

NE

2000

3 h/28 h

42

+

NE

1000

3 h/28 h

43

+

2.0

500

3 h/28 h

64

+

4.0

250

3 h/28 h

75

+a

5.5

125

3 h/28 h

100

+a

1.5

62.5

3 h/28 h

98

-b

NE

31.25

3 h/28 h

84

-

NE

Positive Control

3 h/28 h

67

-

36.1***

 

Negative (vehicle) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 1 µL/mL

Positive control (+S9): Cyclophosphamide, 6 µg/mL

NE: not evaluated

#: compared to the negative (vehicle) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Minimal amount of precipitate/opalescence was observed

b: Discoloured medium

 

*: p<0.05 comparing numbers of aberrant cells excluding gaps with corresponding negative control

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of the test, the test material did not induce chromosome aberrations in the performed experiments with or without metabolic activation. The test material is therefore considered not to be clastogenic in this test system.
Executive summary:

The clastogenic potential of the test material with and without metabolic activation was determined in an in vitro mammalian chromosome aberration test. The study was conducted under GLP conditions and in line with the standardised guidelines OECD 473, EU Method B. 10 and EPA OPPTS 870.5375. Two chromosome aberration assays were performed using Chinese hamster lung fibroblasts (V79) cells. In Assay 1, cells were exposed to the test material for 3 hours with and without metabolic activation and given a 20 hour fixation time. In assay 2, cells were exposed to the test material for 3 hours with and 20 hours without metabolic activation, both were given a fixation time of 28 hours.

Under the conditions of the test, exposure to the test material with or without metabolic activation did not result in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations. Cytotoxicity was observed with and without metabolic activation at concentrations of ≥ 1000 µg/mL and 500 µg/mL, respectively. Therefore, the test material is not considered to be clastogenic in this test system.